Nucleoside diphosphates and triphosphates play important roles in biology. ADP is the immediate precursor for the formation of ATP, the universal currency of cellular energy. GDP is a substrate for succinyl CoA synthetase, a key enzyme of the Krebs cycle, and is formed during gluconeogenesis by phosphoenolpyruvate carboxykinase. It is also essential in G-protein signalling, microtubule growth, and visual excitation. UDP is involved in the epimerisation of galactose to glucose, the formation of sucrose, and in the growth of glycogen. CDP is an important group in the synthesis of phosphoglycerides. Nucleoside diphosphates are also the products of reactions catalysed by several major classes of enzymes, such as triphosphatases and kinases, and are therefore produced by many cellular processes, including motility, muscle contraction, DNA synthesis, transcription, translation and nitrogen fixation.
The detection and measurement of nucleoside diphosphates and triphosphates is thus important in the study of biology and metabolism, particularly in bioenergetics.
Assays for ADP and ATP in biological samples based on luciferase have been known for over 20 years [e.g. refs 1, 2, 3, 4]. Bioluminescent assays for ADP and ATP have been described for use in muscle and adipose tissue biopsies [5] and a three-enzyme bioluminescent system utilising luciferase has been reported for use in bacterial cell extracts [6]. A bioluminescent ADP assay optimised for use at high ATP:ADP ratios has been reported [7], but this requires the enzymatic removal of ATP. In general, it is easier to measure ATP in the presence of ADP than to measure ADP in the presence of ATP.
Enzymatic spectrophotometric assays have also been described [e.g. 8].
Assays for GDP and GTP in biological samples are also well known [e.g. refs 9 & 10].
Reference 11 discloses column-based chromatographic assays for ADP, GDP, CDP and UDP. Radioactive assays for GDP and GTP have also been described [12, 13]. NMR-based assays for measuring in vivo ADP levels are known for yeast [14], and NMR has also been used to measure ADP and ATP and erythrocytes [15].